Methods to determine cell viability or cytotoxicity in response to exposure to a given test agent are key to pharmaceutical and environmental testing, pesticide and herbicide testing, drug discovery, etc. In short, to determine whether a given chemical agent presents a real or potential risk when exposed to a given cell type requires a method that reliably, precisely, and accurately measures cell toxicity and/or viability after exposure to the test agent.
A common method of determining cell viability is based on the ability of the membrane of viable cells to exclude vital dyes such as trypan blue and propidium iodide. Living cells exclude such vital dyes and do not become stained. In contrast, dead or dying cells that have lost membrane integrity allow these dyes to enter the cytoplasm, where the dyes stain various compounds or organelles within the cell.
Non-viable cells that have lost membrane integrity also leak cytoplasmic components into the surrounding medium. Cell death thus can be measured by monitoring the concentration of these cellular components in the surrounding medium. One such method is described in Corey et al. (1997) J. Immunol. Meth. 207:43-51. In this assay, the release of glycerldehyde-3-phosphate dehydrogenase (G3PDH) from dead or damaged cells is measured by coupling the activity of the released G3PDH to the production of ATP.
Other methods to test for cell viability or cell death rely upon the conversion of a dye from one state to another. For example, in a typical format, prior to the reaction the dye absorbs at a first wavelength of radiation. The dye is then converted to a product that absorbs at a second (and different) wavelength of light. By monitoring the conversion of the dye from one state to the other, the extent of cell viability or cell death can be determined. A number of suitable dyes for this purpose are known in the art. The most frequently used of these indicators are electron-acceptor dyes such as tetrazolium salts. Tetrazolium salts known in the prior art include MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), XTT (sodium 3′-{(1-phenylamino-carbonyl)-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate), and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt).
A typical cell viability and proliferation assay using MTS has been described (Buttke et al., (1993) J. Immunol. Methods, 157: 233-240). Dunigan et al. (1995, BioTechniques, 19:640-649) proceed to describe that one of the hallmarks of metabolism is the generation of energy via complex redox reactions of organic molecules. A great many of these reactions utilize β-nicotinamide adenine dinucleotide (NADH) or β-nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donors. While it is theoretically possible to monitor NADH and NADPH concentrations directly via spectrophotometry, from a practical standpoint, direct spectrophotometric analysis is limited due to the presence numerous components that absorb light near the absorption maximum of NADH and NADPH (ε=16,900 at λmax of 259 nm). For example, NAD+, NADP+, DNA, RNA, and most proteins have absorption maxima at approximately 260 nm.
Buttke et al. describe using MTS to measure indirectly the reduction caused by living, proliferating cells, MTS having an absorbance in the visible region when in its reduced form.
The reduced, formazen form of MTS is water soluble and has a broad absorption maximum centered at 450-580 nm. The experiments described by Dunigan et al. are entirely cell free. MTS and MTS/phenazine methosulfate (PMS) solutions were prepared as stock solutions and various combinations of enzymes and reducing agents were added to aliquots of the stock solutions and analyzed spectrophotometrically over time. Various combinations of NADH, NADPH dithiothreitol, 2-mercaptoethanol, malic acid, isocitric acid, malate dehydrogenase, and isocitrate dehydrogenase were tested. The authors found that MTS alone converts only very slowly to its reduced formazen structure. Reactivity of the MTS, however, is hugely accelerated by adding 5% of the electron transfer reagent PMS to the reaction solution. Thus, the authors conclude that MTS/PMS is a useful monitor of NADH and NADPH generation in cell-free aqueous systems.
Lancaster et al., U.S. Pat. No. 5,501,959, issued Mar. 26, 1996, describe a cell is viability and proliferation assay wherein microorganisms, tissue cells, or the like, are incubated in a growth medium in the presence of the dye resazurin and a compound to be tested. A redox stabilizing agent, dubbed a “poising” agent, is also added to the reaction mix to inhibit non-specific autoreduction of the resazurin due to components found within most culture media. In this assay, the resazurin dye is reduced by the activity of living cells. Thus, in the Lancaster et al. assay, the resazurin dye is used as a redox indicator to detect microbial growth, not microbial death. The reduced form of resazurin, known trivially as resorufin, can be detected fluorimetrically or colorimetrically. Resazurin, the oxidized form of the dye, is blue, while resorufin, the reduced form of the dye, is red.
Several cytotoxicity assays can be purchased commercially. For example, Molecular Probes of Eugene, Ore., markets a cytotoxicity assay kit under the trademark “Vybrant”. The “Vybrant”-brand assay detects the release of the cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PDH) from dead and dying cells. This assay detects G6PDH via a two-step process that leads to the reduction of resazurin to resorufin. Molecular Probes+ product literature specifically states that incubations longer than 24 hours “will result in significant degradation of G6PDH, impairing the assay results.” See Molecular Probes' product information flier no. V-23111, revised Oct. 22, 2001. In the hands of the present inventors, however, the half-life of the G6PDH following cell lysis at 37° C. was estimated to be less than two hours at 37° C., thus rendering this kit unsuitable for cytotoxic testing over longer spans of time.
Promega Corporation of Madison, Wisconsin, markets a line of cell viability, cytotoxicity, and cell proliferation assays under the trademarks “CellTiter 96”, “CellTiter-Glo”, and “CytoTox 96.” See Promega Technical Bulletin Nos. 112, 169, 245, and 288. Promega's “CytoTox 96”-brand non-radioactive cytotoxicity assay, for example, is a colorimetric assay for determining the cytotoxicity of a test compound. This assay quantitatively measures the release of LDH from dead cells using an enzyme-linked reduction of INT, an MTS-like tetrazolium dye (see U.S. Pat. No. 5,185,450 for a full description of the MTS dye). This assay is a two-step protocol (i.e., it is non-homogeneous) and the INT dye is detected colorimetrically. This assay uses conditions that are incompatible with living eukaryotic cells as the reaction takes place at a pH of 8.5 with a detergent (Triton-X100) present.
Genotech of St. Louis, Mo., markets a lactate dehydrogenase (LDH)-based cytotoxicity assay under the trademark “CytoScan”. The “CytoScan”-brand assay is a colorimetric method that measures LDH released from dead cells. The LDH released by dead cells is measured via a coupled enzymatic reaction that results in the reduction of a tetrazolium salt into a red-colored formazen. The LDH activity is then determined as a function of NADH oxidation or tetrazolium reduction over a defined period of time. See Genotech catalog no. 786-210. Essentially identical assay kits are marketed by Panvera of Madison, Wis. (LDH Cytotoxicity Detection Kit, Panvera product no. TAK MK401), Oxford Biomedical Research of Oxford, Michigan (Colorimetric Cytotoxicity Assay Kit, Oxford product no. LK 100), and Roche Molecular Biochemicals of Indianapolis, Ind. (Cytotoxicity Detection Kit, Roche catalog no. 1 644 793). All of these kits require a two step procedure to remove culture medium to a separate container and thus are non-homogeneous.
Sigma of St. Louis, Mo., markets two different in vitro toxicology assay kits under the Product Names “Tox-7” and “Tox-8”. The “Tox-7” kit is LDH-based and is essentially identical to Genotech's “CytoScan”-brand assay described in the preceding paragraph. The “Tox-7” assay is a two-step process that requires transferring a supernatant or a cell lysate to a separate vessel, where the supernatant or lysate is then analyzed. Two-step processes are not preferred for high-throughput screening due to the increased material handling requirements. The particular tetrazolium dye used in Sigma's “Tox-7” kit is not specified, but the product literature indicates that the reaction is measured spectrophotometrically at 490 nm, the absorption maximum typical for formazans (and the same wavelength specified in the Genotech product).
All of these commercially available kits require transfer of culture supernatant to an additional vessel for enzymatic measurement of LDH activity and are therefore non-homogeneous.
In Sigma's “Tox-8” assay, bioreduction of resazurin by viable cells (not dead cells) results in the formation of resorufin. The amount of dye reduced can be measured fluorimetrically or spectrophotometrically.
BioSource, International (Camarillo, Calif.) markets kits for measuring cell proliferation and viability under the trademark “alamarBlue,” (see catalog nos. DAL1025 and DALI 100). Like resazurin, “alamarBlue”-brand dye can be used to monitor the reducing environment of living cells. The technology underlying this commercial product is described in Lancaster, U.S. Pat. No. 5,501,959.
The use of absorbent pads impregnated with resazurin and antibiotics for antimicrobial susceptibility testing are described in Baker et al. (1980) Microbiol. 26:248-253 and Canadian Patent No. 1,112,140. Bacterial isolates are applied to the pad in a brain heart infusion broth. The protocols described, however, are not suitable for determining minimum inhibitory concentrations (MIC). Kanazawa et al. (1966) J. Antibiotics 19:229-233 also describe the use of absorbent pads impregnated with resazurin and antimicrobial agents for use in susceptibility testing. Brown et al. (1961) J. Clin. Path. 5:10-13 and U.S. Pat. No. 3,107,204 describe the use of absorbent pads impregnated with a tetrazolium redox indicator and antimicrobial agents, also for use in susceptibility testing.
There remains, however, a long-felt and unmet need for a cytotoxicity assay that measures cell death (rather than viability), wherein the assay is rapid, the components are non-toxic (and thus the test can be run in the presence of the cells being investigated), and wherein the cytoplasmic components measured have a half-life greater than about two hours.